I I DESCRIPTION (provided by applicant): Opioid desensitization following prolonged agonist exposure has been observed in various in vitro preparations, mostly in non-neuronal cells, and implicated in the cellular mechanisms of opioid tolerance. The signaling processes underlying this phenomenon, however, have been poorly understood. In particular, little is known regarding opioid desensitization in neurons with respect to modulation of ion channels. Using voltage-gated Ca 2+ channels (VGCC) as a model system, we examined mu opioid desensitization in murine dorsal root ganglion (DRG) neurons. Our preliminary data showed that brief application of the mu agonist DAMGO inhibited VGCC currents via a G beta gamma-dependent mechanism, whereas prolonged treatments with DAMGO at different time scales resulted in acute or chronic desensitization of current inhibition. The inhibitors of phosphoinositide 3-kinase (PI3K) significantly attenuated DAMGO-induced desensitization, indicating PI3K as a key component of desensitization-related signaling pathways. Further studies using recombinant viral infection techniques showed that the extent of chronic DAMGO desensitization was less pronounced in DRG neurons expressing high levels of mu receptors, and could be further reduced in those expressing a mutant opioid receptor (mu/delta tail chimera) with different phosphorylation and internalization properties. Thus, desensitization in DRG neurons appeared sensitive to changes in receptor density, phosphorylation and internalization. Taken together, we hypothesize that PI3K facilitates opioid desensitization in neurons via activation of several downstream kinase pathways, which may catalyze the phosphorylation of opioid receptors, Go/Gi proteins or Ca2+ channels. Such phosphorylation could rapidly disrupt the receptor-G protein coupling or the G beta gamma-Ca2+ channel interaction, leading to acute desensitization. PI3K cascade-mediated phosphorylation may also trigger long-term changes in receptor recycling and constitutive activity, contributing to chronic desensitization. These hypotheses will be tested in the following specific aims, using primarily electrophysiological approaches combined with biochemical, immunocytochemical and molecular genetic techniques. We will 1) further characterize acute and chronic desensitization of opioid effects on VGCC currents and their modulation by PI3K; 2) identify the signaling pathway(s) involved in PI3K facilitation of opioid desensitization; 3) determine the cellular targets for PI3K cascade-mediate phosphorylation: opioid receptors, G proteins, or Ca2+ channels; 4) investigate the role of PI3K cascades in modulating receptor internalization, recycling and down-regulation; 5) explore the role of receptor constitutive activity in desensitization and PI3K regulation of such activity.